Therapeutic liposome composition and method of preparation

ABSTRACT

Reagents for use in preparing a therapeutic liposome composition sensitized to a target cell are described. The reagents include a liposomal composition composed of pre-formed liposomes having an entrapped therapeutic agent and a plurality of targeting conjugates composed of a lipid, a hydrophilic polymer and a targeting ligand. The therapeutic, target-cell sensitized liposome composition is formed by incubating the liposomal composition with a selected conjugate.

[0001] This application is a continuation of U.S. application Ser. No.09/876,707 filed Jun. 7, 2001, now pending; which is a division of U.S.application Ser. No. 09/517,224 filed Mar. 2, 2000, now U.S. Pat. No.6,316,024; which is a division of U.S. application Ser. No. 09/138,480filed Aug. 21, 1998, now U.S. Pat. No. 6,056,973; which is acontinuation-in-part of U.S. application Ser. No. 08/949,046, filed Oct.10, 1997, now U.S. Pat. No. 5,891,468; which claims the priority of U.S.Provisional Application Serial No. 60/028,269, filed Oct. 11, 1996, nowabandoned. Each of these documents is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to a target-cell sensitizedtherapeutic liposome composition and to a method of preparing thecomposition. A library for preparation of the composition is alsodescribed.

BACKGROUND OF THE INVENTION

[0003] Liposomes, spherical, self-enclosed vesicles composed ofamphipathic lipids, have been widely studied and are employed asvehicles for in vivo administration of therapeutic agents. Inparticular, the so-called long circulating liposomes formulations whichavoid uptake by the organs of the mononuclear phagocyte system,primarily the liver and spleen, have found commercial applicability.Such long-circulating liposomes include a surface coat of flexible watersoluble polymer chains, which act to prevent interaction between theliposome and the plasma components which play a role in liposome uptake.

[0004] More recently, efforts have focused on ways to achieve sitespecific delivery of long-circulating liposomes. In one approach,targeting ligands, such as an antibody, are attached to the liposomes'surfaces. This approach, where the targeting ligand is bound to thepolar head group residues of liposomal lipid components, results ininterference by the surface-grafted polymer chains, inhibiting theinteraction between the bound ligand and its intended target (Klibanov,A. L., et al., Biochim. Biophys. Acta., 1062:142-148 (1991); Hansen, C.B., et al., Biochim. Biophys. Acta, 1239:133-144 (1995)).

[0005] In another approach, the targeting ligand is attached to the freeends of the polymer chains forming the surface coat on the liposomes(Allen. T. M., et al., Biochim. Biophys. Acta, 1237:99-108 (1995);Blume, G., et al., Biochim. Biophys. Acta, 1149:180-184 (1993)). Twoapproaches have been described for preparing a liposome having atargeting ligand attached to the distal end of the surface polymerchains. One approach involves preparation of lipid vesicles whichinclude an end-functionalized lipid-polymer derivative; that is, alipid-polymer conjugate where the free polymer end is reactive or“activated”. Such an activated conjugate is included in the liposomecomposition and the activated polymer ends are reacted with a targetingligand after liposome formation. The disadvantage to this approach isthe difficulty in reacting all of the activated ends with a ligand. Theapproach also requires a subsequent step for separation of the unreactedligand from the liposome composition.

[0006] In another approach, the lipid-polymer-ligand conjugate isincluded in the lipid composition at the time of liposome formation.This approach has the disadvantage that some of the valuable ligandfaces the inner aqueous compartment of the liposome and is unavailablefor interaction with the intended target.

[0007] Both approaches suffer from a lack of flexibility in designing atherapeutic composition that is specific for a target cell for aspecific patient. There is then a need for a liposome composition whichprovides flexibility in choice of the entrapped agent and the targetingligand.

SUMMARY OF THE INVENTION

[0008] Accordingly, it is an object of the invention to provide atherapeutic liposome composition that is readily tailored and designedfor a particular patient.

[0009] It is another object of the invention to provide a kit forformation of a therapeutic, target-cell sensitive liposome composition.

[0010] In one aspect, the invention includes a therapeutic liposomecomposition sensitized to a target cell, comprising (i) a liposomalcomposition composed of pre-formed liposomes having an entrappedtherapeutic agent; and (ii) a plurality of conjugates, each conjugatecomposed of (a) a lipid having a polar head group and a hydrophobictail, (b) a hydrophilic polymer having a proximal end and a distal end,where the polymer is attached at its proximal end to the head group ofthe lipid, and (c) a targeting ligand attached to the distal end of thepolymer. The therapeutic, target-cell sensitized liposome composition isformed by combining the liposomal composition with a conjugate selectedfrom the plurality of conjugates.

[0011] In one embodiment, the targeting ligand is an antibody or anantibody fragment. In one embodiment, the antibody or antibody fragmentis of mouse origin and is humanized to remove murine epitopes.

[0012] In another embodiment, the targeting ligand specifically binds toan extracellular domain of a growth factor receptor. Such receptors areselected from c-erbB-2 protein product of the HER2/neu oncogene,epidermal growth factor receptor, basic fibroblast growth factorreceptor, and vascular endothelial growth factor receptor.

[0013] In another embodiment, the targeting ligand binds a receptorselected from E-selectin receptor, L-selectin receptor, P-selectinreceptor, folate receptor, CD4 receptor, CD19 receptor, αβ integrinreceptors and chemokine receptors.

[0014] The targeting ligand can also be folic acid, pyridoxal phosphate,vitamin B12, sialyl Lewis^(x), transferrin, epidermal growth factor,basic fibroblast growth factor, vascular endothelial growth factor,vascular cell adhesion molecule (VCAM-1), intercellular adhesionmolecule (ICAM-1), platelet endothelial adhesion molecule (PECAM-1), anArg-Gly-Asp (RGD) peptide or an Asp-Gly-Arg (NGR) peptide.

[0015] The hydrophilic polymer surrounding the pre-formed liposomes isselected from the group consisting of polyvinylpyrrolidone,polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide,polymethacrylamide, polydimethylacrylamide,polyhydroxypropylmethacrylate, polyhydroxyethylacrylate,hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol,polyaspartamide and hydrophilic peptide sequences.

[0016] In one embodiment, the hydrophilic polymer is polyethylene glycolof molecular weight between 500-5,000 daltons.

[0017] The entrapped therapeutic agent is, in one embodiment, acytotoxic drug. The drug can be an anthracycline antibiotic selectedfrom doxorubicin, daunorubicin, epirubicin and idarubicin and analogsthereof.

[0018] The cytotoxic agent can also be a platinum compound selected fromcisplatin, carboplatin, ormaplatin, oxaliplatin, zeniplatin, enloplatin,lobaplatin, spiroplatin, ((−)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutane dicarboxylato)platinum),(SP-4-3(R)-1,1-cyclobutane-dicarboxylato(2-)-(2-methyl-1,4-butanediamine-N,N′)platinum),nedaplatin and(bis-acetato-ammine-dichloro-cyclohexylamine-platinum(IV)).

[0019] In another embodiment, the cytotoxic agent is a topoisomerase 1inhibitor selected from the group consisting of topotecan, irinotecan,(7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20(S)-camptothecin),7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin, 9-aminocamptothecinand 9-nitrocamptothecin.

[0020] In another embodiment, the cytotoxic agent is a vinca alkaloidselected from the group consisting of vincristine, vinblastine,vinleurosine, vinrodisine, vinorelbine and vindesine.

[0021] In another embodiment, the entrapped agent is a nucleic acid. Thenucleic acid can be an antisense oligonucleotide or ribozyme or aplasmid containing a therapeutic gene which when internalized by thetarget cells achieves expression of the therapeutic gene to produce atherapeutic gene product.

[0022] In another aspect, the invention includes a plurality oftargeting conjugates for use in preparing a targeted, therapeuticliposome composition. Each conjugate is composed of a (i) a lipid havinga polar head group and a hydrophobic tail, (ii) a hydrophilic polymerhaving a proximal end and a distal end, the polymer attached at itsproximal end to the head group of the lipid, and (iii) a targetingligand attached to the distal end of the polymer.

[0023] The lipid in the conjugates is, in one embodiment, distearoylphosphatidylethanolamine, distearoyl-phosphatidylcholine, monogalactosyldiacylglycerols or digalactosyl diacylglycerols.

[0024] The hydrophilic polymer in the conjugates is selected from thegroup consisting of polyvinylpyrrolidone, polyvinylmethylether,polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropylmethacrylamide, polymethacrylamide,polydimethylacrylamide, polyhydroxypropylmethacrylate,polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose,polyethyleneglycol, polyaspartamide and hydrophilic peptide sequences.

[0025] The targeting ligand of the conjugates can be any of thoserecited above.

[0026] In another aspect, the invention includes a method of formulatinga therapeutic liposome composition having sensitivity to a target cell.The method includes the steps of (i) selecting a liposome formulationcomposed of pre-formed liposomes having an entrapped therapeutic agent;(ii) selecting from a plurality of targeting conjugates a targetingconjugate composed of (a) a lipid having a polar head group and ahydrophobic tail, (b) a hydrophilic polymer having a proximal end and adistal end, where the polymer is attached at its proximal end to thehead group of the lipid, and (c) a targeting ligand attached to thedistal end of the polymer; and (iii) combining the liposome formulationand the selected targeting conjugate to form said therapeutic,target-cell sensitive liposome composition.

[0027] In one embodiment, combining includes incubating under conditionseffective to achieve insertion of the selected targeting conjugate intothe liposomes of the selected liposome formulation.

[0028] In another embodiment, selecting a liposome formulation includesdetermining the sensitivity of the target cell to the therapeuticactivity of the entrapped therapeutic agent.

[0029] In another embodiment, selecting a targeting conjugate includesdetermining the ability of the targeting ligand to bind cell surfacereceptors expressed on the target cell.

[0030] In another embodiment, selecting a targeting conjugate is basedon (i) the ability of a targeting ligand to bind to cell surfacereceptors expressed on the target cell and (ii) the ability of thetarget cell to internalize liposomes bound to the target cell by bindingbetween the target cell and the targeting ligand.

[0031] These and other objects and features of the invention will bemore fully appreciated when the following detailed description of theinvention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 illustrates a library composed of a plurality oftherapeutic pre-formed liposomes and a plurality of targetingconjugates;

[0033]FIGS. 2A-2D are plots showing the fraction of liposomes (peakcentered at fraction 10) and the fraction of micellular targetingconjugates (peak centered at fraction 20) by size exclusionchromatography from samples taken during incubation of a targetingconjugate sialyl-Lewis^(x)-PEG-DSPE with pre-formed liposomes at timesof 0 hours (FIG. 2A), 1 hour (FIG. 2B), 3 hours (FIG. 2C) and 5 hours(FIG. 2D);

[0034]FIG. 3 is a plot showing the time course for insertion of thetargeting conjugate sialyl-Lewis^(x)-PEG-DSPE into pre-formed liposomeswhen incubated at 25° C. (closed circles) and 37° C. (open squares);

[0035]FIG. 4 is a plot showing the blood circulation lifetime oftarget-cell sensitized liposome prepared in accordance with theinvention, where the percent of injected dose in vivo for liposomeshaving E-selectin Fab fragments targeting ligands (30 ligands perliposome represented by solid triangles, 70 ligands per liposomerepresented by solid squares) and for liposomes having a surface coatingof polyethyleneglycol chains (open circles) as a function of time afterdosing; and

[0036]FIGS. 5A-5B are scanned images of micrographs of blood vessels ina window chamber of a mouse dorsal fold, where FIG. 5A is the control ofthe untreated blood vessels under transmitted light, and FIG. 5B is afluorescence micrograph showing binding of fluorsecin-labeled liposomesbearing an E-selectin Fab fragments to endothelial cells in the bloodvessels.

DETAILED DESCRIPTION OF THE INVENTION

[0037] I. Definitions

[0038] Unless otherwise indicated, the terms below have the followingmeaning:

[0039] “Incubating” or “incubating under conditions effective to achieveinsertion” refer to conditions of time, temperature and liposome lipidcomposition which allow for penetration and entry of a selectedcomponent, such as a lipid or lipid conjugate, into the lipid bilayer ofa liposome.

[0040] “Pre-formed liposomes” refers to intact, previously formedunilamellar or multilamellar lipid vesicles.

[0041] “Sensitized to a target cell” or “target-cell sensitized” refersto a liposome which includes a ligand or moiety covalently bound to theliposome and having binding affinity for a receptor expressed on aparticular cell.

[0042] “Therapeutic liposome composition” refers to liposomes whichinclude a therapeutic agent entrapped in the aqueous spaces of theliposomes or in the lipid bilayers of the liposomes.

[0043] “Vesicle-forming lipid” refers to any lipid capable of formingpart of a stable micelle or liposome composition and typically includingone or two hydrophobic acyl hydrocarbon chains or a steroid group andmay contain a chemically reactive group, such as an amine, acid, ester,aldehyde or alcohol, at its polar head group.

[0044] II. Liposome and Conjugate Library

[0045] In one aspect, the invention includes a kit or “library” forpreparation of a therapeutic, target-cell sensitized liposomecomposition. FIG. 1 shows such a library 10, where a plurality 12 oftargeting conjugates 12(a), 12(b), 12(c), etc. and a plurality 14 ofpre-formed therapeutic liposome compositions, such compositions 14(a),14(b), 14(c) are shown. The targeting conjugates and pre-formed liposomepluralities are shown in suspension form in vials ready for use, howeverit will be appreciated that other storage forms are contemplated, suchas lyophilized or freeze-dried.

[0046] The targeting conjugates in the library are lipid-polymer-ligandconjugates and will be described in more detail below. The conjugates inthe library differ in the targeting ligand attached to thelipid-polymer, as well as in the lipid and polymer components. Exemplaryligands and lipid and polymer components will be set forth below.

[0047] The pre-formed liposomes in the library are either conventionalliposomes containing an entrapped therapeutic agent or are liposomeshaving a surface coating of hydrophilic polymer chains, as will bedescribed below. The pre-formed liposomes in the library differ from oneanother generally in the entrapped therapeutic agent and exemplaryagents will be set forth below. The pre-formed liposomes can also differfrom one another in the liposome lipid components.

[0048] A therapeutic, target-cell sensitized liposome composition isprepared from the library as follows. A composition specific for asubject suffering from a particular condition, for example a solid tumorof the lung, a bacterial infection or a viral infection, is prepared byselecting a targeting conjugate from the library. The targetingconjugate is selected either according to knowledge of those of skill inthe art of ligand-receptor binding pairs or by obtaining a suitablepatient sample, e.g., a fluid sample, a biopsy or the like. The sampleis tested by means known to those in the art for expression of a varietyof receptors to determine the appropriate targeting ligand.

[0049] A pre-formed therapeutic liposome composition is selected basedon knowledge of those of skill in the art of the therapeutic agentsappropriate for treatment of the particular condition. Alternatively,the therapeutic liposome composition is selected after performingchemosensitivity tests to determine the effect of the entrapped agent oncells of concern obtained from the patient biopsy or fluid sample.

[0050] Following selection of the targeting conjugate and of thepre-formed liposome composition, the target-cell sensitized, therapeuticliposome composition for the subject is prepared by combining the twocomponents. As will be described, the components are combined underconditions effective to achieve insertion of the targeting conjugateinto the liposome bilayer to create the target-cell sensitizedliposomes. After insertion is complete, the composition is administeredto patient.

[0051] The therapeutic pre-formed liposomes and the targeting conjugatewill now be described in more detail.

[0052] A. Therapeutic Pre-formed Liposome Component

[0053] As discussed above, one component of the kit or library forpreparing the composition of the invention is a plurality of pre-formedliposomes having an entrapped therapeutic or diagnostic agent. In thissection, the liposome lipid components, exemplary agents and methods ofpreparing the liposomes are described.

[0054] 1. Liposome Components

[0055] Liposomes suitable for use in the composition of the presentinvention include those composed primarily of vesicle-forming lipids.Such a vesicle-forming lipid is one which (a) can form spontaneouslyinto bilayer vesicles in water, as exemplified by the phospholipids, or(b) is stably incorporated into lipid bilayers, with its hydrophobicmoiety in contact with the interior, hydrophobic region of the bilayermembrane, and its head group moiety oriented toward the exterior, polarsurface of the membrane.

[0056] The vesicle-forming lipids of this type are preferably oneshaving two hydrocarbon chains, typically acyl chains, and a head group,either polar or nonpolar. There are a variety of syntheticvesicle-forming lipids and naturally-occurring vesicle-forming lipids,including the phospholipids, such as phosphatidylcholine,phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, andsphingomyelin, where the two hydrocarbon chains are typically betweenabout 14-22 carbon atoms in length, and have varying degrees ofunsaturation. The above-described lipids and phospholipids whose acylchains have varying degrees of saturation can be obtained commerciallyor prepared according to published methods. Other suitable lipidsinclude glycolipids, cerebrosides and sterols, such as cholesterol.

[0057] Cationic lipids are also suitable for use in the liposomes of theinvention, where the cationic lipid can be included as a minor componentof the lipid composition or as a major or sole component. Such cationiclipids typically have a lipophilic moiety, such as a sterol, an acyl ordiacyl chain, and where the lipid has an overall net positive charge.Preferably, the head group of the lipid carries the positive charge.Exemplary cationic lipids include 1,2-dioleyloxy-3-(trimethylamino)propane (DOTAP);N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammoniumbromide (DMRIE); N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);3β[N-(N′,N′-dimethylaminoethane) carbamoly] cholesterol (DC-Chol); anddimethyldioctadecylammonium (DDAB).

[0058] The cationic vesicle-forming lipid may also be a neutral lipid,such as dioleoylphosphatidyl ethanolamine (DOPE) or an amphipathiclipid, such as a phospholipid, derivatized with a cationic lipid, suchas polylysine or other polyamine lipids. For example, the neutral lipid(DOPE) can be derivatized with polylysine to form a cationic lipid.

[0059] In another embodiment, the vesicle-forming lipid is selected toachieve a specified degree of fluidity or rigidity, to control thestability of the liposome in serum, to control the conditions effectivefor insertion of the targeting conjugate, as will be described, and tocontrol the rate of release of the entrapped agent in the liposome.

[0060] Liposomes having a more rigid lipid bilayer, or a liquidcrystalline bilayer, are achieved by incorporation of a relatively rigidlipid, e.g., a lipid having a relatively high phase transitiontemperature, e.g., up to 60° C. Rigid, i.e., saturated, lipidscontribute to greater membrane rigidity in the lipid bilayer. Otherlipid components, such as cholesterol, are also known to contribute tomembrane rigidity in lipid bilayer structures.

[0061] On the other hand, lipid fluidity is achieved by incorporation ofa relatively fluid lipid, typically one having a lipid phase with arelatively low liquid to liquid-crystalline phase transitiontemperature, e.g., at or below room temperature.

[0062] As will be described below, the targeted, therapeutic liposomecomposition of the invention is prepared using pre-formed liposomes anda targeting conjugate, which are incubated together under conditionseffective to achieve insertion of the conjugate into the liposomebilayer. More specifically, the two components are incubated togetherunder conditions which achieve insertion of the conjugate in such a waythat the targeting ligand is oriented outwardly from the liposomesurface, and therefore available for interaction with its cognatereceptor.

[0063] Vesicle-forming lipids having phase transition temperatures fromapproximately 2° C.-80° C. are suitable for use in the pre-formedliposome component of the present composition. By way of example, thelipid distearyl phosphatidylcholine (DSPC) has a phase transitiontemperature of 62° C. and the lipid hydrogenated soy phosphatidylcholine(HSPC) has a phase transition temperature of 58° C. Phase transitiontemperatures of many lipids are tabulated in a variety of sources, suchas Avanti Polar Lipids catalogue and Lipid Thermotropic Phase TransitionDatabase (LIPIDAT, NIST Standard Reference Database 34).

[0064] In one embodiment of the invention, a vesicle-forming lipidhaving a phase transition temperature between about 30-70° C. isemployed. In another embodiment, the lipid used in forming the liposomesis one having a phase transition temperature within about 20° C., morepreferably 10° C., most preferably 5° C., of the temperature to whichthe ligand in the targeting conjugate can be heated without affectingits binding activity.

[0065] It will be appreciated that the conditions effective to achieveinsertion of the targeting conjugate into the liposome are determinedbased on several variables, including, the desired rate of insertion,where a higher incubation temperature may achieve a faster rate ofinsertion, the temperature to which the ligand can be safely heatedwithout affecting its activity, and to a lesser degree the phasetransition temperature of the lipids and the lipid composition. It willalso be appreciated that insertion can be varied by the presence ofsolvents, such as amphipathic solvents including polyethyleneglycol andethanol, or detergents.

[0066] In one embodiment of the invention, the pre-formed liposomes alsoinclude a vesicle-forming lipid derivatized with a hydrophilic polymer.As has been described, for example in U.S. Pat. No. 5,013,556, includingsuch a derivatized lipid in the liposome composition forms a surfacecoating of hydrophilic polymer chains around the liposome. The surfacecoating of hydrophilic polymer chains is effective to increase the invivo blood circulation lifetime of the liposomes when compared toliposomes lacking such a coating.

[0067] Vesicle-forming lipids suitable for derivatization with ahydrophilic polymer include any of those lipids listed above, and, inparticular phospholipids, such as distearoyl phosphatidylethanolamine(DSPE).

[0068] Hydrophilic polymers suitable for derivatization with avesicle-forming lipid include polyvinylpyrrolidone,polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide,polymethacrylamide, polydimethylacrylamide,polyhydroxypropylmethacrylate, polyhydroxyethylacrylate,hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol,polyaspartamide and hydrophilic peptide sequences. The polymers may beemployed as homopolymers or as block or random copolymers.

[0069] A preferred hydrophilic polymer chain is polyethyleneglycol(PEG), preferably as a PEG chain having a molecular weight between500-10,000 daltons, more preferably between 1,000-5,000 daltons. Methoxyor ethoxy-capped analogues of PEG are also preferred hydrophilicpolymers, commercially available in a variety of polymer sizes, e.g.,120-20,000 daltons.

[0070] Preparation of vesicle-forming lipids derivatized withhydrophilic polymers has been described, for example in U.S. Pat. No.5,395,619. Preparation of liposomes including such derivatized lipidshas also been described, where typically, between 1-20 mole percent ofsuch a derivatized lipid is included in the liposome formulation.

[0071] 2. Therapeutic Agent

[0072] The pre-formed liposomes include an agent entrapped in theliposome. Entrapped is intended to include encapsulation of an agent inthe aqueous core and aqueous spaces of liposomes as well as entrapmentof an agent in the lipid bilayer(s) of the liposomes.

[0073] Agents contemplated for use in the composition of the inventionare widely varied, and include both therapeutic applications and thosefor use in diagnostic applications.

[0074] Therapeutic agents include natural and synthetic compounds havingthe following therapeutic activities: anti-arthritic, anti-arrhythmic,anti-bacterial, anticholinergic, anticoagulant, antidiuretic, antidote,antiepileptic, antifungal, anti-inflammatory, antimetabolic,antimigraine, antineoplastic, antiparasitic, antipyretic, antiseizure,antisera, antispasmodic, analgesic, anesthetic, beta-blocking,biological response modifying, bone metabolism regulating,cardiovascular, diuretic, enzymatic, fertility enhancing,growth-promoting, hemostatic, hormonal, hormonal suppressing,hypercalcemic alleviating, hypocalcemic alleviating, hypoglycemicalleviating, hyperglycemic alleviating, immunosuppressive,immunoenhancing, muscle relaxing, neurotransmitting,parasympathomimetic, sympathominetric plasma extending, plasmaexpanding, psychotropic, thrombolytic and vasodilating.

[0075] In a preferred embodiment, the entrapped agent is a cytotoxicdrug, that is, a drug having a deleterious or toxic effect on cells.Exemplary cytotoxic agents include the anthracycline antibiotics such asdoxorubicin, daunorubicin, epirubicin and idarubicin, and analogs ofthese, such as epirubidin and mitoxantrone; platinum compounds, such ascisplatin, carboplatin, ormaplatin, oxaliplatin, zeniplatin, enloplatin,lobaplatin, spiroplatin, ((−)-(R)-2- aminomethylpyrrolidine(1,1-cyclobutane dicarboxylato)platinum) (DWA2114R),(SP-4-3(R)-1,1-cyclobutane-dicarboxylato(2-)-(2-methyl-1,4-butanediamine-N,N′)platinum)(CI-973), nedaplatin (254-S) and(bis-acetato-ammine-dichloro-cyclohexylamine-platinum(IV)) (JM-216)(Weiss, R. B., et al., Drugs, 46(3):360-377 (1993)); and vincaalkaloids, such as vincristine, vinblastine, vinleurosine, vinrodisine,vinorelbine (navelbine) and vindesine.

[0076] Another preferred group of cytotoxic agents is a topoisomerase Iinhibitor, such as camptothecin and its analogues, including SN-38((+)-(4S)-4,11-diethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7]-indolizino[1,2-b]quinoline-3,14(4H,12H)-dione);9-aminocamptothecin; 9-nitrocamptothecin, topotecan (hycamtin;9-dimethyl-aminomethyl-10-hydroxycamptothecin); irinotecan (CPT-11;7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxy-camptothecin),which is hydrolyzed in vivo to SN-38); 7-ethylcamptothecin and itsderivatives (Sawada, S. et al., Chem. Pharm. Bull., 41(2):310-313(1993)); 7-chloromethyl-10,11-methylene-dioxy-camptothecin; and others(SN-22, Kunimoto, T. et al., J. Pharmacobiodyn., 10(3):148-151: (1987);DX-8951f and GG-211((7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20(S)-camptothecin))(Rothenberg, M. L., Ann. Oncol., 8(9):837-855 (1997)), and7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin (Chong Kun Dang Corp.,Seoul Dorea, CKD602).

[0077] In another embodiment, the entrapped therapeutic agent is anangiogenesis inhibitor, such as angiostatin, endostatin and TNFα.

[0078] In another embodiment, the entrapped therapeutic agent in anucleic acid, selected from a variety of DNA and RNA based nucleicacids, including fragments and analogues of these. A variety of genesfor treatment of various conditions have been described, and codingsequences for specific genes of interest can be retrieved from DNAsequence databanks, such as GenBank or EMBL. For example,polynucleotides for treatment of viral, malignant and inflammatorydiseases and conditions, such as, cystic fibrosis, adenosine deaminasedeficiency and AIDS, have been described. Treatment of cancers byadministration of tumor suppressor genes, such as APC, DPC4, NF-1, NF-2,MTS1, RB, p53, WT1, BRCA1, BRCA2 and VHL, are contemplated.

[0079] Administration of the following nucleic acids for treatment ofthe indicated conditions are also contemplated: HLA-B7, tumors,colorectal carcinoma, melanoma; IL-2, cancers, especially breast cancer,lung cancer, and tumors; IL-4, cancer; TNF, cancer; IGF-1 antisense,brain tumors; IFN, neuroblastoma; GM-CSF, renal cell carcinoma; MDR-1,cancer, especially advanced cancer, breast and ovarian cancers; and HSVthymidine kinase, brain tumors, head and neck tumors, mesothelioma,ovarian cancer.

[0080] The polynucleotide can be an antisense DNA oligonucleotidecomposed of sequences complementary to its target, usually a messengerRNA (mRNA) or an mRNA precursor. The mRNA contains genetic informationin the functional, or sense, orientation and binding of the antisenseoligonucleotide inactivates the intended mRNA and prevents itstranslation into protein. Such antisense molecules are determined basedon biochemical experiments showing that proteins are translated fromspecific RNAs and once the sequence of the RNA is known, an antisensemolecule that will bind to it through complementary Watson-Crick basepairs can be designed. Such antisense molecules typically containbetween 10-30 base pairs, more preferably between 10-25, and mostpreferably between 15-20.

[0081] The antisense oligonucleotide can be modified for improvedresistance to nuclease hydrolysis, and such analogues includephosphorothioate, methylphosphonate, phosphodiester and p-ethoxyoligonucleotides (WO 97/07784).

[0082] The entrapped agent can also be a ribozyme or catalytic RNA.

[0083] 3. Liposome Preparation

[0084] The liposomes may be prepared by a variety of techniques, such asthose detailed in Szoka, F., Jr., et al., Ann. Rev. Biophys. Bioeng.9:467 (1980), and specific examples of liposomes prepared in support ofthe present invention will be described below. Typically, the liposomesare multilamellar vesicles (MLVs), which can be formed by simplelipid-film hydration techniques. In this procedure, a mixture ofliposome-forming lipids of the type detailed above dissolved in asuitable organic solvent is evaporated in a vessel to form a thin film,which is then covered by an aqueous medium. The lipid film hydrates toform MLVs, typically with sizes between about 0.1 to 10 microns.

[0085] As described above, in one embodiment, the pre-formed liposomesinclude a vesicle-forming lipid derivatized with a hydrophilic polymerto form a surface coating of hydrophilic polymer chains on the liposomessurface. Such a coating is preferably prepared by including between 1-20mole percent of the derivatized lipid with the remaining liposomeforming components, e.g., vesicle-forming lipids. Exemplary methods ofpreparing derivatized lipids and of forming polymer-coated liposomeshave been described in co-owned U.S. Pat. Nos. 5,013,556, 5,631,018 and5,395,619, which are incorporated herein by reference. It will beappreciated that the hydrophilic polymer may be stably coupled to thelipid, or coupled through an unstable linkage which allows the coatedliposomes to shed the coating of polymer chains as they circulate in thebloodstream or in response to a stimulus.

[0086] The therapeutic or diagnostic agent of choice can be incorporatedinto liposomes by standard methods, including (i) passive entrapment ofa water-soluble compound by hydrating a lipid film with an aqueoussolution of the agent, (ii) passive entrapment of a lipophilic compoundby hydrating a lipid film containing the agent, and (iii) loading anionizable drug against an inside/outside liposome pH gradient. Othermethods, such as reverse evaporation phase liposome preparation, arealso suitable.

[0087] Polynucleotides, oligonucleotides, other nucleic acids, such as aDNA plasmid, can be entrapped in the liposome by condensing the nucleicacid in single-molecule form. The nucleic acid is suspended in anaqueous medium containing protamine sulfate, spermine, spermidine,histone, lysine, mixtures thereof, or other suitable polycationiccondensing agent, under conditions effective to condense the nucleicacid into small particles. The solution of condensed nucleic acidmolecules is used to rehydrate a dried lipid film to form liposomes withthe condensed nucleic acid in entrapped form. A similar approach tocondensing nucleic acids for entrapment in liposomes is described inco-pending U.S. patent application Ser. No. 09/103,341.

[0088] The pre-formed liposomes of the invention are preferably preparedto have substantially homogeneous sizes in a selected size range,typically between about 0.01 to 0.5 microns, more preferably between0.03-0.40 microns. One effective sizing method for REVs and MLVsinvolves extruding an aqueous suspension of the liposomes through aseries of polycarbonate membranes having a selected uniform pore size inthe range of 0.03 to 0.2 micron, typically 0.05, 0.08, 0.1, or 0.2microns. The pore size of the membrane corresponds roughly to thelargest sizes of liposomes produced by extrusion through that membrane,particularly where the preparation is extruded two or more times throughthe same membrane. Homogenization methods are also useful fordown-sizing liposomes to sizes of 100 nm or less (Martin, F. J., inSPECIALIZED DRUG DELIVERY SYSTEMS-MANUFACTURING AND PRODUCTIONTECHNOLOGY, (P. Tyle, Ed.) Marcel Dekker, N.Y. pp. 267-316 (1990)).

[0089] B. Targeting Conjugates

[0090] The kit or library of the invention also includes a targetingconjugate, now to be described. The targeting conjugate is composed of(i) a lipid having a polar head group and a hydrophobic tail, e.g., avesicle-forming lipid and any of those described above are suitable;(ii) a hydrophilic polymer attached to the head group of thevesicle-forming lipid, and any of the polymers recited above aresuitable; and (iii) a targeting ligand attached to the polymer.

[0091] The targeting ligand for use in the conjugate can be selectedfrom a wide variety of moieties capable of targeting the pre-formedliposomes to a selected cell or tissue. Examples of suitable ligandssuitable are listed in Table 1. TABLE 1 LIGAND-RECEPTOR PAIRS ANDASSOCIATED TARGET CELL LIGAND RECEPTOR CELL TYPE Folate folate receptorepithelial carcinomas, bone marrow stem cells water soluble vitaminsvitamin receptor various cells Pyridoxyl phosphate CD4 CD4+ lymphocytesApolipoproteins LDL liver hepatocytes, vascular endothelial cellsInsulin insulin receptor pancreatic islet cells Transferrin Transferrinreceptor endothelial cells (brain) Galactose Asialoglycoprotein liverhepatocytes receptor Sialyl-Lewis^(x) E, P selectin activatedendothelial cells Mac-1 L selectin neutrophils, leukocytes VEGF Flk-1,2tumor epithelial cells basic FGF FGF receptor tumor epithelial cells EGFEGF receptor epithelial cells VCAM-1 α₄β₁ integrin vascular endothelialcells ICAM-1 α_(L)β₂ integrin vascular endothelial cells PECAM-1/CD31α_(ν)β₃ integrin vascular endothelial cells Fibronectin α_(ν)β₃ integrinactivated platelets Osteopontin α_(ν)β₁ and α_(ν)β₅ endothelial cellsand integrins smooth muscle cells in atherosclerotic plaques RGDpeptides and α_(ν)β₃ integrin tumor endothelial cells, peptide mimetics(i.e. vascular smooth muscle amino acid sequences cells of matrixproteins) HIV GP 120/41 or CD4 CD4+lymphocytes GP120 C4 domain peptomersHIV GP120/41 (Macro- Chemokine receptor macrophages, dendritic phagetropic isolates) CC-CRK-5 cells Anti-cell surface receptor Cell surfaceerythrocytes, platelets, antibodies (or receptors endothelial cells,fragments thereof), lymphocytes, tumors such as anit-HER2/neu,anti-selectin, anti- VEGF Anti-cell surface receptor Cell surface bonemarrow stem cells, antibodies (or receptors such as malignant B and Tcells fragments thereof) CD34, CD19, CD4, CD7, CD8, CD20, CD22

[0092] One preferred ligand is an antibody or an antibody fragment. Itwill be appreciated that the antibody or antibody fragment can be ofmouse origin and humanized to remove murine surface recognitionfeatures.

[0093] In another preferred embodiment, the targeting ligand binds to anextracellular domain of a growth factor receptor. Exemplary receptorsinclude the c-erbB-2 protein product of the HER2/neu oncogene, epidermalgrowth factor (EGF) receptor, basic fibroblast growth receptor (basicFGF) receptor and vascular endothelial growth factor receptor, E-, L-and P-selectin receptors, folate receptor, CD4 receptor, CD19 receptor,αβ integrin receptors and chemokine receptors.

[0094] 1. Preparation of Targeting Conjugates

[0095] As described above, the targeting ligand is covalently attachedto the free distal end of the hydrophilic polymer chain, which isattached at its proximal end to a vesicle-forming lipid. There are awide variety of techniques for attaching a selected hydrophilic polymerto a selected lipid and activating the free, unattached end of thepolymer for reaction with a selected ligand, and in particular, thehydrophilic polymer polyethyleneglycol (PEG) has been widely studied(Allen, T. M., et al., Biochemicia et Biophysica Acta 1217:99-108(1995); Zalipsky, S., Bioconjugate Chem., 4(4):296-299 (1993); Zalipsky,S., et al., FEBS Lett. 353:71-74 (1994); Zalipsky, S., et al.,Bioconjugate Chemistry, 705-708 (1995); Zalipsky, S., in STEALTHLIPOSOMES (D. Lasic and F. Martin, Eds.) Chapter 9, CRC Press, BocaRaton, Fla. (1995)).

[0096] Generally, the PEG chains are functionalized to contain reactivegroups suitable for coupling with, for example, sulfhydryls, aminogroups, and aldehydes or ketones (typically derived from mild oxidationof carbohydrate portions of an antibody) present in a wide variety ofligands (see Table 1). Examples of such PEG-terminal reactive groupsinclude maleimide (for reaction with sulfhydryl groups),N-hydroxysuccinimide (NHS) or NHS-carbonate ester (for reaction withprimary amines), hydrazide or hydrazine (for reaction with aldehydes orketones), iodoacetyl (preferentially reactive with sulfhydryl groups)and dithiopyridine (thiol-reactive). Synthetic reaction schemes foractivating PEG with such groups are set forth in U.S. Pat. Nos.5,631,018, 5,527,528, 5,395,619, and the relevant sections describingsynthetic reaction procedures are expressly incorporated herein byreference.

[0097] It will be appreciated that any of the hydrophilic polymersrecited above in combination with any of the vesicle-forming lipidsrecited above can be employed for the targeting conjugate and suitablereaction sequences can be determined by those of skill in the art.

[0098] III. Preparation of the Liposome Composition

[0099] The section above described preparation of the components in thelibrary of the invention, namely the pre-formed liposomes and thetargeting conjugates. This section describes preparation of thetarget-cell sensitized, therapeutic liposome composition using these twocomponents.

[0100] As discussed briefly above, a pre-formed therapeutic liposomecomposition and a targeting conjugate are selected from the library. Thetwo components are combined under conditions effective to achieveinsertion of the targeting conjugate into the liposome lipid bilayer toform the target-cell sensitized composition.

[0101] In studies performed in support of the invention, a targetingconjugate of the ligand sialyl-Lewis^(x) was attached to PEG-DSPEaccording to known methods (DeFrees, S. A., et al., J. Am. Chem. Soc.,118:6101-6104 (1996)). Sialyl-Lewis^(x) can be used to target liposomesto cells expressing endothelial leukocyte adhesion molecule-1 (ELAM-1 orE-selectin) for delivery of a therapeutic agent to a site ofinflammation. ELAM-1 is expressed on the surface of endothelial cells ofblood vessels adjacent to sites of inflammation. ELAM-1 recognizes andbinds the polysaccharide moiety sialyl-Lewis^(x) which is present onsurfaces of neutrophils, and recruits neutrophils to sites ofinflammation.

[0102] Pre-formed liposomes were prepared as described in Example 1 andwere composed of partially hydrogenated soy-bean phosphatidylcholine(PHPC), cholesterol and mPEG-DSPE in a molar ratio of 55:40:3. Theliposomes were sized to a diameter of about 100 nm. The liposomes wereincubated at 37° C. with 1.2 mole percent sialyl-Lewis^(x)-PEG-DSPEtargeting conjugate to achieve insertion of the conjugate into thepre-formed liposomes.

[0103] Insertion of the conjugate into the liposomes was monitored bysampling the mixture and tracking the relative amounts of micellularconjugate and liposomes by size exclusion chromatography, and theresults are shown in FIGS. 2A-2D.

[0104] In FIGS. 2A-2D, the liposome fraction of the incubation mixtureis represented by the peak centered around fraction 10 and themicellular targeting conjugate is represented by the peak centeredaround fraction 20. FIG. 2A shows the initial composition mixture, attime zero and FIGS. 2B-2D show the composition after 1, 3 and 5 hoursincubation, respectively. Disappearance as a function of incubation timeof the conjugate micelles (peak at fraction 20) is apparent, indicatinginsertion of the targeting conjugate into the pre-formed liposomes.

[0105]FIG. 3 shows the time-course of insertion ofsialyl-Lewis^(x)-PEG-DSPE targeting conjugate into PHPC:Chol:mPEG-DSPE(55:40:3) pre-formed liposomes at 37° C. (open squares) and 25° C.(closed circles). Insertion of the conjugate into the pre-formedliposomes proceeded more rapidly at 37° C., however insertion at ambienttemperature was also substantial.

[0106] Other experiments in support of the invention were performedusing the targeting conjugate YIGSRG-PDG-DSPE, prepared as described inZalipsky, S., et al., Bioconjugate Chemistry, 8(2):111-118 (1997). Thepentapeptide YIGSRG (Tyr-Ile-Gly-Ser-Arg) is the shortest fragment ofthe basement membrane glycoprotein laminin which retains bindingactivity to laminin cell surface receptors. In these studies,essentially the same time course of insertion was observed (data notshown) upon incubation with pre-formed liposomes ofPHPC:cholesterol:mPEG-DSPE.

[0107] Table 2 shows the average particle size determined by dynamiclight scattering after a 5 hour incubation period at 37° C. ofpre-formed liposomes and a targeting conjugate, either YIGSRG-PEG-DSPEor sialyl-Lewis^(x)-PEG-DSPE. The average particle size after incubationand insertion of the targeting conjugate increased only slightly. TABLE2 Liposome Size Targeting Conjugate Before insertion After insertionYIGSRG-PEG-DSPE 100 nm 105 nm Sialyl-Lewis^(x)-PEG-DSPE  98 nm  99 nm

[0108] The studies above illustrate preparation of a target-cellsensitized liposome composition by incubating pre-formed liposomes witha ligand-polymer-lipid targeting conjugate. It will be appreciated thatliposomes having any composition and any selected entrapped therapeuticagent can be used in conjunction with the desired targeting conjugate.The ligand-polymer-lipid conjugate readily inserts into pre-formedliposomes in a time and temperature dependent fashion, and, as will beappreciated, is variable according to the liposome and ligandcompositions.

[0109] IV. In Vivo Administration of the Composition

[0110] Liposomes having an E-selectin Fab fragment targeting ligand wereprepared in accordance with the invention for in vivo administration torodents. As described in Example 2, an anti-E-selectin Fab fragment wasconjugated to PEG-DSPE to form an E-selectin Fab-PEG-DSPE targetingconjugate. The targeting conjugate was incubated with pre-formed¹¹¹In-labelled-liposomes composed of partially hydrogenated soyphosphatidylcholine (PHPC), PEG-DSPE and cholesterol in a 55:40:3 molarratio in an amount sufficient to obtain 12, 20, 33, 40 and 70 Fabresidues per 100 nm liposome (Example 2B). The insertion procedureresulted in greater than 95% of the targeting conjugates being insertedinto the pre-formed liposomes. In one embodiment of the invention, theinsertion efficiency is greater than 90%, more preferably greater than95%.

[0111] The liposomes containing 30 Fab residues per liposome and 70 Fabresidues per liposome were administered to rats to determine the bloodcirculation lifetime of the liposomes. As a control,¹¹¹In-labelled-liposomes of PHPC, cholesterol and PEG-DSPE (molar ratioof 55:40:3) were administered. The results are shown in FIG. 4, wherethe liposomes having 70 Fab residues per liposome (solid squares) and 30Fab residues per liposomes (solid triangles) have a pharmacokineticprofile similar to that of the control liposomes (open circles). Asseen, 24 hours after administration, nearly 25% of the injected doseremains in circulation in the bloodstream.

[0112] As described in Example 2C, pre-formed liposomes composed ofhydrogenated soy phosphatidylcholine (HSPC), cholesterol, PEG-DSPE andfluorescein-labelled DHPE, in a molar ratio of 53.5/40/4/2.5, wereincubated with the E-selectin-PEG-DSPE targeting conjugate at 37° C. for1 hour. The fluorescein-labeled liposomes were administered to miceequipped with a dorsal skin fold window chamber. Endotoxin was appliedtopically in the window chamber 10 minutes after intravenous injectionof the liposomes. FIGS. 5A-5B are scanned images of photomicrographs ofthe blood vessels under transmitted light prior to liposomeadministration (FIG. 5A) and 5 hours after administration of thetarget-cell sensitized, fluorescein-labeled liposomes (FIG. 5B).

[0113] As can be seen in FIG. 5B, the E-selectin Fab liposomes targetthe endothelial cells along the blood vessels. The appearance ofE-selectin antigen peak was around 5 hours after endotoxin treatment,indicating that the binding activity of the E-selectin antibody wasretained.

[0114] V. Method of Using the Library

[0115] In accordance with the invention, a plurality of targetingconjugates and a plurality of liposome formulations with a variety ofentrapped therapeutic agents are available for selection according tothe indication to be treated. In this section, preparation and use ofthe library will be further demonstrated by describing suitable librarycomponents for treatment of an exemplary indication, breast cancer.

[0116] For treatment of human breast cancer, the library of theinvention includes a plurality of targeting conjugates in the form ofpre-filled vials containing the conjugate as a purified, sterilemicellar suspension in an appropriate buffer. The plurality of targetingconjugates can include the following.

[0117]1. Anti c-erbB-2-PEG-DSPE

[0118] The c-erbB-2 receptor of the HER2-neu oncogene is over-expressedin many human breast cancer cells. Humanized monoclonal antibodies havebeen developed which bind with high affinity to the c-erbB-2 receptor(Baselga J., et al., J Clin Oncol., 14(3):737-44 (1996)). Single chainsFv fragments of the anti c-erbB-2 C6.5 antibody into which a terminalcysteine group is inserted are obtained as described by Schier et al.(Immunotechnology, 1(1):73-81 (1995)). The whole c-erbB-2 antibody isconjugated to PEG-DSPE having a reactive hydrazide moiety. The sFvfragment containing the terminal cysteine (and thus a free thiol group)is conjugated to PEG-DSPE-maleimide, under conditions like thosedescribed for the conjugation of the anti-E selected Fab′ antibodyfragment to the same compound in Example 2.

[0119]2. anti-EGFR-PEG-DSPE Targeting Conjugate

[0120] Epidermal Growth Factor Receptor (EGFR) and a deletion-mutantform of EGFR (EGFRvIII) are over-expressed in certain breast cancers,gliomas and lung tumors (Beckmann, M. W., Geburtshilfe Frauenheilkd,55(5):258-65 (1995)). Whole mouse monoclonal antibodies which bind thisreceptor are obtained as described by Wikstrand et al (Cancer Res.,55(14):3140-8 (1995)). These whole antibodies are conjugated to DSPE-PEGhaving an active hydrazide end as has been described in the art.

[0121] It will be appreciated that other antibody or antibody fragmentswhich are known to bind receptors over-expressed in breast cancers cellsincluding integrins such as a_(v)B₅ and interlukin-8 are available andcan be linked to PEG-DSPE for preparation of targeting conjuates for usein the library.

[0122] 3. PEG-DSPE Targeting Conjugate Including a_(v) Integrin-bindingRGD Peptides

[0123] Proteins that contain the Arg-Gly-Asp (RGD) attachment site,together with the integrins that serve as receptors for them, constitutea major recognition system for cell adhesion. The RGD sequence is thecell attachment site for proliferating vascular endothelial cells whichform the blood supply to tumors (during angiogenesis). Such attachmentsare mediated by a_(v), integrins expressed by these endothelial cells.The integrin-binding activity of matrix adhesion proteins can bereproduced by short synthetic peptides containing the RGD sequence.Reagents that bind selectively to only one or a few of the RGD-directedintegrins can be designed by cyclizing peptides with selected sequencesaround the RGD and by synthesizing RGD mimics. Such RGD peptides can beisolated by using phage display peptide libraries (Pasqualini, R., andRuoslahti, E., Nature, 380(6572):364-6 (1996)). Two of thesepeptides—one containing an a_(v) integrin-binding Arg-Gly-Asp motif andthe other an Asn-Gly-Arg motif—have been identified that bindselectively to tumor vasculature. These can be linked to liposomes usingthe methods described herein.

[0124] As can be appreciated, a plurality of other PEG-DSPE conjugatesof ligands, such as folate or transferin, which may bind to receptors onhuman breast cancer cells are prepared according to the examples setherein and by methods known in the art.

[0125] Continuing with the example of using the library for treatment ofhuman breast cancer, the library further includes a therapeutic liposomecomposition or a plurality of liposome compositions containingencapsulated agents appropriate for treating human breast cancer cellsin vivo. The pre-formed liposomes are in the form of pre-filled vialscontaining the liposomes as a sterile suspension in appropriate buffersis created. Liposome containing the following entrapped agents areexemplary for the human breast cancer example: doxorubicin, cisplatin,water-soluble camptothecin derivatives (e.g. topotecan, navelbine,vincristine, antisense oligonucleotides, p53 gene, HSVtk gene, aradiation sensitizer and an angiogenesis inhibitor.

[0126] To use the library, a targeting conjugate and a therapeuticliposome composition are selected. Selection of the targeting conjugateis based upon the expression of the conjugate's cognate receptor onindividual patient's breast cancer cells. For example, it is common totest for the expression of a variety of receptors on cancer cellsobtained from patients during biopsy. Clinical reference laboratoriesroutinely screen biopsy specimens for estrogen receptor status andc-erbB-2 expression status is becoming routine with the clinicaldevelopment of HERCEPTIN an anti-tumor therapeutic antibody productdescribed by Baselga, et al, (J. Clin Oncol., (3):737-44 (1996)).Exemplary methods for determining c-erbB-2 receptor status are given bySjogren, et al. (J Clin Oncol., 16(2):462-9 (1998)). Patients whosetumors overexpress c-erbB-2 receptor are identified by this approach.EGFR receptor status is determined by similar methodology (Newby, J. C.et al., Br J Cancer., 71(6):1237-42 (1995)). Expression of otherreceptors is determined by similar methodology.

[0127] Next, a pre-formed therapeutic liposome composition is selectedfrom the library. A variety of methods exist to screen for thesensitivity of breast cancer cells taken at biopsy to the cell killingeffects of drugs in vitro and in vivo (chemosensitivity testing) andexemplary methods are described by Tomikawa, et al. (Anticancer Res.,18(2A):1059-62 (1998)) and by Coley, et al. (Anticancer Res.17(1A):231-6 (1997)) and by Andreotti, et al. (Cancer Res.,55(22):5276-82 (1995)). In vitro cytotoxicity is often expressed as theconcentration of a particular cancer drug needed to inhibit cancer cellproliferation by 50% in culture (IC₅₀). In a typical screening test,cells obtained from a patient's biopsy specimen are teased apart(mechanically and/or by enzyme treatment), suspended in a medium whichsupports their growth and placed in wells of a culture plate. Drugs atvarious dilutions are added and any growth inhibition of the cellscaused by the drug is measured. IC₅₀ values are derived from thesemeasurements. Drugs that kill the cells or inhibit growth atconcentrations at or below IC₅₀ values that can be achieved in vivo areconsidered as candidates for therapeutic intervention.

[0128] In an alternative approach, a therapeutic agent can be selectedon the basis of historical information and accepted clinical practice(see for example, Handbook of Cancer Chemotherapy, ₃rd edition, R. T.Skeell, editor, A, Little Brown, Boston, 1991, pp 77-138.). For example,doxorubicin is known to be one of the most active agents against humanbreast cancer. Therefore, in a plurality of liposome-encapsulated cancerdrugs, doxorubicin would represent an obvious selection for thetreatment of breast cancer based on accepted clinical practice.

[0129] After selection of the targeting conjugate and the therapeuticpre-formed liposome composition, the two reagents are combined to createtarget cell-sensitized therapeutic liposome composition tailored to anindividual patient's cancer. The contents of the vial containing theconjugate and the vial containing the pre-formed therapeutic liposomecomposition, selected as described above and based upon the expressionof the appropriate cell surface receptor and the sensitivity of the cellto growth inhibitory action of the encapsulated agent, are combinedunder the conditions described effective to achieve insertion of theconjugate into the liposome bilayer. Aseptic technique is used,preferably in a hospital pharmacy or other appropriate setting. Once thetarget-sensitized therapeutic liposome composition is formed, it isadministered to the patient for which it was created. The liposomes aretypically in suspension form and are administered parenterally,preferably intravenously. Other routes of administration are suitable,including subcutaneous, intramuscular, interlesional (to tumors),intertracheal by inhalation, topical, internasal, intraocular, viadirect injection into organs and intravenous.

[0130] As can be appreciated, tailoring the formulation in this way tothe individual patient maximizes the likelihood of therapeutic benefitprovided by the targeting component and the encapsulated drug.

[0131] It will be appreciated that the dosage will depend on theliposome composition and the condition to be treated. Suitable dosagescan be readily determined by those of skill in the art.

[0132] Use of the library will now be demonstrated for treatment of apatient suffering from a hematological disease, e.g. a B-cell or T-cellmalignancy, such as B-cell leukemias/lymphomas, multiple myeloma ,T-cell lymphoma and acute lymphocytic leukemia.

[0133] As described above, the library includes a plurality of targetingconjugates. Targeting conjugates suitable for selection includelipid-polymer-antibody conjugates, where the antibody is a monoclonalantibody or antibody fragment having a specific recognition to a B-cellor a T-cell epitope, as has been described in U.S. Pat. No. 5,620,689,which is incorporated herein by reference. For example, the antibody canbe one that recognizes the B-cell epitopes CD19, CD20, CD22 or CD77. Theantibody can be one that recognizes the T-cell epitopes CD4, CD7 or CD8.

[0134] The library further includes liposomes having entrapped agents.For treatment of hematological disorders, liposomes having the followingentrapped agents are potential candidates for selection from thelibrary: doxorubicin, vincristine, lomustine, interferon, melphalan,cyclophosphamide, prednisone, chlorambucil, carmustin and dexamethasone.

[0135] A blood or tissue sample is taken from the patient suffering fromthe hematological disorder for determination of the expression ofvarious receptors, such as CD19, CD20, CD22, CD4, CD7, CD8. If theorigin of the disorder is known to be either B-cell or T-cell, thereceptor screening can of course be more selective, e.g., if thedisorder is B-cell related, then the sample can be tested for expressionof CD19, CD20 and CD22. Based on the results of the screening, asuitable targeting conjugate is chosen.

[0136] A therapeutic agent for treatment of the disorder is selectedfrom the library using the procedures described in the breast cancerexample above.

[0137] The selected conjugate and liposome composition are incubatedtogether as described above to form the target-cell sensitized,therapeutic liposome composition specific for the patient. Suitabledosages for the composition can be initially based on the standardchemotherapeutic dose and adjusted accordingly over the course oftreatment by monitoring the disease progression.

EXAMPLES

[0138] The following examples illustrate methods of preparing thecomposition of the present invention. The examples are in no wayintended to limit the scope of the invention.

Example 1 Preparation of Pre-formed Liposomes and Insertion of TargetingConjugate

[0139] Liposomes were prepared by mixing partially hydrogenated soy-beanphosphatiylcholine (PHPC, iodine value of 35, Lipoid (Ludwigshafen,Germany)), cholesterol (Croda (Fullerton, Calif.)) and mPEG-DSPE(prepared as described in Zalipsky, S., et al., Bioconjugate Chemistry,4:296-299 (1993)) at a molar ratio of 55:40:3 in chloroform and/ormethanol in a round bottom flask. The solvents were removed by rotaryevaporation, and the dried lipid film produced was hydrated with eithersodium phosphate buffer (10 mM, 140 mM NaCl, pH 7) or HEPES buffer (25mM, 150 mM NaCl, pH 7) to produce large multilamellar vesicles. Theresulting vesicles were passed repeatedly under pressure through 0.2,0.1 and 0.05 μm pore size polycarbonate membranes, until the averagesize distribution for the diameter (monitored by dynamic lightscattering using a Coulter N4MD (Hialeah, Fla.)) was approximately 100nm. The mean particle diameter measured from 12 different batches rangedform 92 to 111 nm with an average 98 nm.

[0140] Targeting conjugates of and YIGSRG-PEG-DSPE were preparedaccording to Zalipsky, S., et al., Bioconjugate Chemistry, 8(2):111-118(1997).

[0141] The pre-formed liposomes were incubated at either 25° C. or 37°C. with 1.2 mole percent of one of the targeting conjugates. At varioustime points, targeting conjugates (micelles) were separated frominserted targeting conjugates (liposomes) by size exclusionchromatography. For the sialyl-Lewis^(x)-PEG-DSPE conjugate, a BiogelA50M column equilibrated with 10 mM sodium phosphate, 140 mM sodiumchloride, and 0.02% NaN₃ at pH 6.5 was used. For YIGSRG-PEG-DSPEconjugate, a Sepharose 4B column was used with 10% sucrose and 10 mMHEPES at pH 7.0 as eluent. The results for the sialyl-Lewis^(x)-PEG-DSPEconjugate are shown in FIGS. 2A-2D for the 0, 1, 3 and 5 hour timepoints, where the peak centered around fraction 10 corresponds to theliposomes and the peak centered around fraction 20 corresponds to themicellular, targeting conjugate.

[0142] The collected fractions (1 mL) from the size exclusionchromatograph were diluted 1:10 in methanol, and analyzed for ligandcontent by HPLC (Shimadzu and Rainin systems), with the results shown inFIG. 3.

Example 2 Preparation of Anti-E-selectin Fab Conjugate and Insertioninto Pre-formed Liposomes

[0143] A. Preparation of the Targeting Conjugate

[0144] An anti-E-selectin Fab fragment was conjugated to PEG-DSPE toform a targeting conjugate as follows. An aqueous solution of 750 mM2-mercaptoethylamine as a reducing agent was prepared. 10 μl of themercaptoethylamine was added to 1 ml of 5 mg/ml anti E-selectin Fabfragment in 50 mM sodium acetate and 125 mM NaCl, pH=5.0. The finalconcentration of reducing agent was 7.5 mM. The solution was incubatedat 37° C. for 30 minutes. The excess reducing agent was removed on a10DG-column (Bio-Rad) equilibrated with 25 mM HEPES/0.9% saline buffer.The collected fractions were analyzed spectrophotometrically todetermine the fractions containing the Fab fragments. These fractionswere pooled and diluted 1:50 in phosphate buffered saline to determinethe protein concentration.

[0145] The Fab fragments (molecular weight of 3,000 Daltons) were mixedin a 1:1 molar ratio with PEG-DSPE (molecular weight 50,000 daltons)having an active maleimide end group (prepared as described in U.S. Pat.No. 5,527,528). The two components were incubated overnight at roomtemperature. The unreacted maleimide was quenched with 2 mMβ-mercaptoethanol for 30 minutes at room temperature. The free Fabfragments and β-mercaptoethanol were separated from the Fab-PEG-DSPEconjugate on an S-200 column equilibrated in 25 mM HEPES/0.9%saline atpH 7.2. Fractions of 1 ml were collected and read on thespectrophotometer at 280 nm to determine the fractions containing theconjugate and the free Fab fragments. The fractions were pooledaccordingly and the concentration of the Fab-PEG-DSPE micellularsolution is determined spectrophotometrically (280 nm). The efficiencyof conjugation of the Fab fragment to the maleimide-PEG-DSPE wasapproximately 40%.

[0146] B. Insertion of the Conjugate into Pre-formed Liposomes

[0147] Liposomes of partially hydrogenated soy phosphatidylcholine(PHPC), PEG-DSPE and cholesterol in a 55:40:3 molar ratio were preparedas described in Example 1. Depending on the desired number of targetingligands per liposomes, an amount of the Fab-PEG-DSPE conjugate was addedto a suspension of liposomes and incubated overnight at roomtemperature. A 100 μl aliquot of the insertion mixture was taken andplaced on a SEPHAROSE 4B column (0.7×30 cm) to separate the free Fabconjugate from the liposomes. 1 ml fractions were collected and read onthe spectrophotometer to determine the amount of conjugate inserted intothe pre-formed liposomes. Greater than 95% of the conjugates wereinserted into the pre-formed liposomes.

[0148] Following insertion of the targeting conjugate, an aliquiot ofthe liposomes were analyzed by amino acid analysis to determine theprotein concentration. Another aliquot was analyzed for phosphoruscontent. Based on these values, the amount of protein per liposome wasdetermined.

[0149] Using this insertion procedure, liposomes containing 12, 20, 33,40 and 70 Fab residues per 100 nm liposome, as determined by amino acidanalysis, were prepared. ¹¹¹In-labelled-liposomes containing 30 Fabresidues per liposome and 70 Fab residues per liposome were administeredto rats to determine the blood circulation lifetime of the liposomes. Asa control, ¹¹¹In-labelled-liposomes of PHPC, cholesterol and PEG-DSPE(molar ratio of 55:40:3) were administered. The results are shown inFIG. 4.

[0150] C. In Vivo Targeting

[0151] E-selectin Fab-PEG-DSPE targeting conjugate was inserted intopre-formed liposomes as follows. The pre-formed liposomes were composedof hydrogenated soy phosphatidylcholine (HSPC), cholesterol and PEG-DSPEin a molar ratio of 53.5/40/4. The liposomes included 2.5 mole percentof the lipid marker of fluorescein-DHPE (Molecular Probes, Inc.). Thepre-formed liposomes were incubated with the micellular solution of thetargeting conjugate at 37° C. for 1 hour. The insertion mixture wasplaced on a Bio-Rad A50m column equilibrated with 25 mM HEPES/saline pH7.2 and 0.5 ml fractions were collected. Spectrophotometric analysis ofthe fractions indicated that the insertion efficiency of the Fabtargeting conjugate into the liposomes was approximately 100% after 2hours at 37° C.

[0152] The fluorescein-labeled liposomes were administered to miceequipped with a window chamber in a dorsal skin fold. Endotoxin wasapplied topically in the window chamber 10 minutes after intravenousinjection of the liposomes. FIGS. 5A-5B are photomicrographs (scannedimages) of the blood vessels under transmitted light prior to liposomeadministration (FIG. 5A) and 5 hours after administration of thetarget-cell sensitized, fluorescein-labeled liposomes (FIG. 5B).

[0153] Although the invention has been described with respect toparticular embodiments, it will be apparent to those skilled in the artthat various changes and modifications can be made without departingfrom the invention.

1. A composition comprising reagents for use in preparing a therapeuticliposome composition sensitized to a target cell, said reagentscomprised of (a) a liposomal composition composed of pre-formedliposomes having an entrapped therapeutic agent; and (b) a plurality ofconjugates, each conjugate composed of (i) a lipid having a polar headgroup and a hydrophobic tail, (ii) a hydrophilic polymer having aproximal end and a distal end, said polymer attached at its proximal endto the head group of the lipid, and (iii) a targeting ligand attached tothe distal end of the polymer; wherein said reagents are mixed to formthe therapeutic, target-cell sensitized liposome composition.
 2. Thecomposition of claim 1, wherein the targeting ligand is an antibody oran antibody fragment.
 3. The composition of claim 2, wherein theantibody or antibody fragment is a humanized murine antibody.
 4. Thecomposition of claim 2, wherein the targeting ligand specifically bindsto an extracellular domain of a growth factor receptor.
 5. Thecomposition of claim 4, wherein the receptors are selected from thegroup consisting of c-erbB-2 protein product of the HER2/neu oncogene,epidermal growth factor receptor, basic fibroblast growth factorreceptor, and vascular endothelial growth factor receptor.
 6. Thecomposition of claim 2, wherein the targeting ligand binds a receptorselected from the group consisting of E-selectin receptor, L-selectinreceptor, P-selectin receptor, folate receptor, CD4 receptor, CD19receptor, _(αβ) integrin receptors and chemokine receptors.
 7. Thecomposition of claim 1, wherein the targeting ligand is selected fromthe group consisting of folic acid, pyridoxal phosphate, vitamin B12,sialyl Lewis^(x), transferrin, epidermal growth factor, basic fibroblastgrowth factor, vascular endothelial growth factor, VCAM-1, ICAM-1,PECAM-1, RGD peptides and NGR peptides.
 8. The composition of claim 1,wherein the targeting ligand binds a receptor on a malignant B-cell orT-cell, said receptor selected from the group consisting of CD19, CD20,CD22, CD4, CD7 and CD8.
 9. The composition of claim 1, wherein thehydrophilic polymer is selected from the group consisting ofpolyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline,polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropylmethacrylamide, polymethacrylamide,polydimethylacrylamide, polyhydroxypropylmethacrylate,polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose,polyethyleneglycol, polyaspartamide and hydrophilic peptide sequences.10. The composition of claim 1, wherein the hydrophilic polymer ispolyethylene glycol.
 11. The composition of claim 10, wherein thepolyethylene glycol has a molecular weight between 500-5,000 daltons.12. The composition of claim 1, wherein the liposomes further contain acationic lipid.
 13. The composition of claim 1, wherein the entrappedtherapeutic agent is a cytotoxic drug.
 14. The composition of claim 13,wherein the cytotoxic drug is an anthracycline antibiotic selected fromthe group consisting of doxorubicin, daunorubicin, epirubicin andidarubicin and analogs thereof.
 15. The composition of claim 13, whereinthe cytotoxic agent is a platinum compound selected from cisplatin,carboplatin, ormaplatin, oxaliplatin, zeniplatin, enloplatin,lobaplatin, spiroplatin, ((−)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutane dicarboxylato)platinum),(SP-4-3(R)-1,1-cyclobutane-dicarboxylato(2-)-(2-methyl-1,4-butanediamine-N,N′)platinum),nedaplatin and(bis-acetato-ammine-dichloro-cyclohexylamine-platinum(IV)).
 16. Thecomposition of claim 13, wherein the cytotoxic agent is a topoisomerase1 inhibitor selected from the group consisting of topotecan, irinotecan,(7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20(S)-camptothecin),7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin, 9-aminocamptothecinand 9-nitrocamptothecin.
 17. The composition of claim 13, wherein thecytotoxic agent is a vinca alkaloid selected from the group consistingof vincristine, vinblastine, vinleurosine, vinrodisine, vinorelbine andvindesine.
 18. The composition of claim 1, wherein the entrapped agentis a nucleic acid.
 19. The composition of claim 18, wherein the nucleicacid is an antisense oligonucleotide or ribozyme.
 20. The composition ofclaim 18, wherein the nucleic acid is a plasmid containing a therapeuticgene which when internalized by the target cells achieves expression ofthe therapeutic gene to produce a therapeutic gene product. 21-56(Canceled)
 57. A therapeutic liposome composition having sensitivity toa target cell, comprising pre-formed liposomes having an entrappedtherapeutic agent; and one or more conjugates composed of (i) a lipidhaving a polar head group and a hydrophobic tail, (ii) a hydrophilicpolymer having a proximal end and a distal end, said polymer attached atits proximal end to the head group of the lipid, and (iii) a targetingligand attached to the distal end of the polymer; wherein saidpre-formed liposomes and said one or more conjugates are in individualcontainers prior to formation of the therapeutic liposome compositionhaving sensitivity to a target cell.
 58. The composition of claim 57,wherein said conjugate is comprised of a vesicle-forming lipid,polyethylene glycol, and a targeting ligand having binding affinity fora cell surface receptor.
 59. The composition of claim 57, wherein saidpre-formed liposomes include as the entrapped therapeutic agentdoxorubicin.
 60. The composition of claim 58, wherein said pre-formedliposomes include as the entrapped therapeutic agent doxorubicin.